Chỉnh sửa gen thụ thể CD163 ở tế bào nguyên bào sợi của lợn bằng công nghệ CRISPR/CAS9

Nguyễn Văn Ba, Giang Thị Thanh Nhàn, Nguyễn Thị Lệ Hương, Phạm Thu Thảo, Nguyễn Khánh Vân và Phạm Doãn Lân.

Abstract

Editting CD163 recetor gene in porcine fibroblast by using CRISPR/CAS9 system
The CD163 receptor plays an important role in the biology of porcine reproductive and
respiratory syndrome virus (PRRSV) by allowing the virus to enter and infect target cells. This
study was carried out to edit the exon7- CD163 of pig fibroblasts to create a pig resistant to pathogenic
porcine reproductive and respiratory syndrome virus. The CRISPR pX459 vector was attached a gRNA
specific to the exon 7 of the CD163 receptor and transformed into E. coli. The vector CRISPR pX459-
CD163 was successfully cloned and isolated at a high concentration (1,700 ng/µl). The CRISPR
pX459-CD163 vector was transfected into porcine fibroblast cells in medium supplemented with
7,5µl Lipofectamine TM 3000 and incubated for 48 hours. Use media containing the antibiotic
Puromycin at concentrations from 2 to 10 µg/ml to screen successfully transfected cells. As a result,
a porcine fibroblast cell line was edited in exon 7 of the CD163 gene carrying C-to-T nucleotide
change, resulting in an Alanine–to-Valine substitution. The research results have opened up the
prospect of applying the combination of CRISPR/CAS9 system and cloning to create pigs resistant
to pathogenic porcine reproductive and respiratory syndrome virus